Abstract. F-spondin is a member of a family of proteins that collectively belong to a subgroup of TSR (thrombospondin) type I class molecules. We have discovered that F-spondin expression is significantly increased in osteoarthritic cartilage as well as in rodent meniscectomy models of OA. Preliminary studies indicate that F-spondin has significant effects on human chondrocyte metabolism and is also expressed in the hypertrophic regions of embryonic growth plates where it acts to regulate mineralization and endochondral bone formation. This proposal is designed to characterize the functional effects of F-spondin, which have major and previously unrecognized implications for the progression of OA. We will test two central hypotheses: 1) F-spondin modulates collagen degradation via unrecognized pathways that include activation of TGF- and induction of MMPs and 2) F-spondin induces hypertrophic differentiation of articular chondrocytes and plays an essential role in the regulation of mineralization and endochondral bone formation. The specific aims designed to test these hypotheses are as follows: In SPECIFIC AIM 1 we will investigate the effects of F-spondin on the regulation of hypertrophy and mineralizing activity in human articular chondrocytes in vitro culture models. Cartilage specimens will be examined histologically to investigate the link between F-spondin and other characteristic hypertrophic/ossification markers of OA chondrocytes. In SPECIFIC AIM 2 we will investigate the molecular mechanism(s) of F-spondin-mediated collagen degradation in OA cartilage. Explant or cell cultures will be used to a) identify MMPs induced by F- spondin and examine their role in F-spondin-mediated collagen degradation b) establish the role of TGF- in modulation of F-spondin functions and c) compare functional activity of the full length F-spondin molecule relative to its proteolytic fragments. In SPECIFIC AIM 3 we will identify and characterize the interacting proteins of F-spondin. a) investigate the interaction of F-spondin with the Latency-associated peptide (LAP) of the latent TGF- complex and b) utilize yeast 2 hybrid and proteomic technologies to identify novel F- spondin binding proteins (proteases, receptors, matrix molecules) that may regulate its activity in articular cartilage. In SPECIFIC AIM 4 we will investigate the expression and function of F-spondin in cartilage in vivo. We will investigate F-spondin expression in cartilage in vivo i) during endochondral bone development and ii) in the mouse meniscectomy model of OA. We will generate an F-spondin knockout mouse and characterize the changes in cartilage phenotype during endochondral bone development and OA disease progression. Understanding the regulation of chondrocyte functions by F-spondin could lead to novel strategies for cartilage repair and disease modifying treatments for osteoarthritis.